**4. Typical complete methods**

Some typical methods are given completely to make the understanding of the process of aflatoxin analysis.

#### **4.1 Determination of aflatoxin M1 with fluorometer**

This is very simple and efficient method. It is also a specific method. The analysis is carried out with Fluorometer along with the use of affinity chromatography columns for clean-up step according to the method described by Hansen (1990). Before analysis, sample is brought to room temperature. To remove cream from the milk sample, it is centrifuged at 2000×*g* for 10 minutes. The 10 mL sample of skim milk is passed through AflaTest affinity column of the Vicam, USA. These affinity columns contain antibodies to aflatoxin. The column is then washed twice with 10 mL portions of 10% methanol and the aflatoxin M1 is eluted from the affinity column by passing 1.0 mL of 80% methanol. All the sample eluate (1.0 mL) is collected in a glass cuvette.

A sample enzyme-linked immunosorbent assay calibration curve is shown in the Fig. 2. The ELISA reader gives absorbance readings from which % absorbance is calculated. For standard solutions, the % absorbance is plotted against aflatoxin concentration to get the calibration curve. The aflatoxin concentration is on x-axis and % absorbance is on y-axis.

y = -1104.1x + 94.857

= 0.9907

R2

0 0.02 0.04 0.06 0.08 0.1

**AFM1 Concentration (microgram/Kg)**

Some typical methods are given completely to make the understanding of the process of

This is very simple and efficient method. It is also a specific method. The analysis is carried out with Fluorometer along with the use of affinity chromatography columns for clean-up step according to the method described by Hansen (1990). Before analysis, sample is brought to room temperature. To remove cream from the milk sample, it is centrifuged at 2000×*g* for 10 minutes. The 10 mL sample of skim milk is passed through AflaTest affinity column of the Vicam, USA. These affinity columns contain antibodies to aflatoxin. The column is then washed twice with 10 mL portions of 10% methanol and the aflatoxin M1 is eluted from the affinity column by passing 1.0 mL of 80% methanol. All the sample eluate

From the calibration curve, aflatoxin concentration is calculated for samples.

Fig. 2. A sample enzyme-linked immunosorbent assay calibration curve

0

**4. Typical complete methods** 

(1.0 mL) is collected in a glass cuvette.

**4.1 Determination of aflatoxin M1 with fluorometer** 

aflatoxin analysis.

20

40

60

**% Absorbance**

80

100

120

The concentration of aflatoxin M1 is measured in a fluorometer (Vicam, USA) with the option of 360 nm excitation filter and 440 nm emission filter. The results are recorded using digital Fluorometer readout with automatic printing device.

#### **4.2 Determination of aflatoxin M1 by HPLC**

A very competent method used for determination of aflatoxin M1 is that of the AOAC Official Method 2000.08 (AOAC Official Method 2000.08, 2005). Details of the method are given in coming lines.

#### **4.2.1 Extraction procedure**

After warming at about 37oC in water bath, liquid milk is centrifuged at 2000×*g* to separate fat layer and then filtered. The prepared test portion of 50 mL is transferred into syringe barrel attached with immonoaffinity column (IAC) and passed at slow steady flow rate of 2- 3 mL/ min. The washing of column is done with 20 mL water and then it is blown to dryness and afterwards aflatoxin M1 is eluted with 4 mL pure acetonitrile by allowing it to be in contact with the column at least 60 seconds. The eluate is evaporated to dryness using gentle stream of nitrogen and at the time of LC (liquid chromatography) determination it is diluted with the mobile phase.

#### **4.2.2 LC Determination with fluorescence detection**

The HPLC system of Agilent 1100 series (Agilent, USA), equipped with an auto sampler LAS G1313A and a fluorescence detector FLD G1321A with excitation and emission wavelength of 365nm and 435nm respectively, may be used for aflatoxin M1 determination. Any other suitable system may be used instead of the above mentioned system. The ZORBAX Eclipse XDB-C18 (Octadecyl silane chemically bonded to porous silica) column (Agilent, USA), 4.6×150 mm with particle size 5 µm in diameter, may be used. Acetonitrile in ratio of 25% with 75% water is used as mobile phase. The flow rate is 0.8 mL/min. Calibration curve is determined using a series of calibration solutions of aflatoxin M1 in acetonitrile. The concentrations of calibration curves may be in the range of 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 µg/ L. The retention time for aflatoxin M1 may be in the range of 6.1-6.5 min.

#### **4.2.3 Calculations**

Calculations are made according to the following equation:

$$\mathbf{W\_m = W\_a \times (V\_t/V\_i) \times (1/V\_s)}$$

Where Wm = amount of aflatoxin M1 in the test sample in µg/L; Wa = amount of aflatoxin M1 corresponding to area of aflatoxin M1 peak of the test extract (ng); Vf = the final volume of re-dissolved eluate (µL); Vi = volume of injected eluate (µL); Vs = volume of test portion (milk) passing through the column (mL).

#### **4.3 Determination of aflatoxin B1 by HPLC**

An important method for the determination of aflatoxin B1 is that of the AOAC Official Method 994.08 which has been described here with small modifications (AOAC Official Method 994.08, 2000).

Aflatoxin Measurement and Analysis 143

An ELISA method for the determination of aflatoxin M1 in cheese is given here as described by the protocol provided with RIDASCREEN® ELISA kit (RIDASCREEN® Aflatoxin M1

Cheese (2.0g) samples are first of all triturated. Extraction is completed with 40 mL dichloromethane by shaking for 15 minutes. The suspension is filtered and 10 mL of the extract is evaporated at 60°C under weak N2-stream. The oily residue is re-dissolved in 0.5 mL methanol, 0.5 mL PBS buffer and 1 mL n-heptane. After mixing thoroughly, it is centrifuged for 15 minutes at 2700 × *g*. The upper heptane layer is removed completely. From the lower methanolic-aqueous phase, 100µL is taken and diluted with 400 mL buffer 1

The standard solutions (100µL) and prepared samples (100µL) are added into the microtiter well placed in the microwell holder. Gentle mixing is accomplished by shaking the plate manually and incubated for 30 minutes at room temperature (20-25°C) in the dark. The liquid is poured out of the wells and microwell holder is tapped vigorously upside down against adsorbent paper to ensure complete removal of liquid from the wells. The wells are washed by adding 250 µL washing buffer in each well and poured out the liquid again. Washing step is repeated for two times. Then 100 µL of the diluted enzyme conjugate is added and mixed gently by shaking the plate manually and incubated for 15 minutes at room temperature in the dark. After incubation the wells are washed again. The 100 µl of substrate/chromogen is added and mixed gently by shaking the plate manually and incubated for 15 minutes at room temperature in the dark. Now stop solution (100µL) is added in each well. Mixing is done by shaking the plate manually. The absorbance is measured photometrically at 450 nm against an air blank with in 15 minutes after the

(Absorbance of standard or sample / absorbance of zero standard) × 100 = % absorbance The zero standard is made equal to 100 % and absorbance values are taken in percentages. A calibration curve is obtained by plotting %absorbance values for the standards against the aflatoxin M1 concentration (µg/L). The concentration of aflatoxin M1 in samples is

The methods of measurement and analysis of aflatoxins have been discussed in this chapter. Some photographs were taken by the author himself, while others were downloaded from internet. Some analytical studies in the aflatoxin analysis have been included to have the insight of methods' application and their development. Typical complete methods have been included as exemplary methods, so the understanding of the process of aflatoxin

**4.4 Determination of aflatoxin M1 in cheese by ELISA** 

30/15, 2007).

**4.4.1 Sample preparation** 

**4.4.2 Test procedure** 

addition of stop solution.

calculated from the calibration curve.

The following formula is used to measure the % absorbance.

**4.4.3 Calculations** 

**5. Conclusion** 

analysis may become clear.

and 100µL of it is used per well in the test.

#### **4.3.1 Extraction and clean-up procedure**

A test portion of 50.0g and 100mL extraction solvent (850mL acetonitrile with 150mL deionized water) is taken in 250mL Erlenmeyer flask and placed in a shaker for 1 hour at high speed. After filtration, 8mL extract is taken with pipette in 10mL glass tube. MycoSep® column (rubber flange end) is pushed slowly into the tube. As column is pushed into the tube, extract is forced through frit, through 1-way valve, and through packing material and is collected in column reservoir. The purified extract (2mL) is transferred quantitatively from top of column to screw cap vial (derivatization vial) and is evaporated under nitrogen.

#### **4.3.2 Aflatoxin derivatization**

After adding n-hexane (200µL) in the derivatization vial to re-dissolve aflatoxin, 50µL of trifluoroacetic acid is added and it is mixed on vortex mixer for 30 seconds. After five minutes, 1.95mL of deionized water: acetonitrile (9:1) mixture is added and again mixed on vortex mixer for 30 seconds. Layers are allowed to separate and aqueous layer (lower layer) containing aflatoxins is removed, filtered through 0.45µm syringe filter and then injected onto LC column.

#### **4.3.3 LC Determination with fluorescence detection**

The high-performance liquid chromatography equipment (LC-10, Shimadzu, Japan), comprising liquid pump LC-10AS, column oven CTO-10A, system controller SCL-10A, fluorescence detector RF-530, communication bus module CBM-101, and data acquisition software class LC-10A may be used for aflatoxin B1 determination. Any other suitable system may also be used instead of the above said one. The excitation wavelength of 365nm and emission wavelength of 435nm is set during analysis. The stainless steel column Discovery® C-18 of Supelco (Bellifonte, PA, USA) with dimensions of 25cm×4.6mm (id) and with particle size of 5 µm diameter may be used. The mobile phase (acetonitrile: methanol: deionized water in the ratio of 20:20:60) is degassed with sonicator before use. The flow rate is 1.0 mL/ min. Calibration curve is determined using a series of calibration solutions of aflatoxin B1 in acetonitrile. The concentrations of calibration solutions may be 0.5, 1.5, 2.5, 5.0, 10.0, and 15.0 µg/ L. The retention time for aflatoxin B1 is near to 5.36 minutes or may be slightly different by changing conditions or instrument.

#### **4.3.4 Calculations**

Aflatoxin B1 peak is identified in derivatized extract chromatogram by comparing its retention time with corresponding peak in the standard chromatogram. The quantity of the aflatoxin B1, '*C'* is determined in the derivatized extract (injected) from the respective standard curves. The concentration of aflatoxin B1 is calculated in test sample as follows:

Aflatoxins B1 ng/g = *C*/*W*

$$\text{W} = 50 \text{g} \times (2 \text{mL} / \text{ } \text{200mL}) \times (0.02 \text{mL} / \text{ } \text{2mL}) = 0.005 \text{g}$$

Where *W* = equivalent weight of test portion (in 20µL) injected into LC; *C* = ng aflatoxin (in 20µL) injected into LC.

A test portion of 50.0g and 100mL extraction solvent (850mL acetonitrile with 150mL deionized water) is taken in 250mL Erlenmeyer flask and placed in a shaker for 1 hour at high speed. After filtration, 8mL extract is taken with pipette in 10mL glass tube. MycoSep® column (rubber flange end) is pushed slowly into the tube. As column is pushed into the tube, extract is forced through frit, through 1-way valve, and through packing material and is collected in column reservoir. The purified extract (2mL) is transferred quantitatively from top of column to screw cap vial (derivatization vial) and is

After adding n-hexane (200µL) in the derivatization vial to re-dissolve aflatoxin, 50µL of trifluoroacetic acid is added and it is mixed on vortex mixer for 30 seconds. After five minutes, 1.95mL of deionized water: acetonitrile (9:1) mixture is added and again mixed on vortex mixer for 30 seconds. Layers are allowed to separate and aqueous layer (lower layer) containing aflatoxins is removed, filtered through 0.45µm syringe filter and then injected

The high-performance liquid chromatography equipment (LC-10, Shimadzu, Japan), comprising liquid pump LC-10AS, column oven CTO-10A, system controller SCL-10A, fluorescence detector RF-530, communication bus module CBM-101, and data acquisition software class LC-10A may be used for aflatoxin B1 determination. Any other suitable system may also be used instead of the above said one. The excitation wavelength of 365nm and emission wavelength of 435nm is set during analysis. The stainless steel column Discovery® C-18 of Supelco (Bellifonte, PA, USA) with dimensions of 25cm×4.6mm (id) and with particle size of 5 µm diameter may be used. The mobile phase (acetonitrile: methanol: deionized water in the ratio of 20:20:60) is degassed with sonicator before use. The flow rate is 1.0 mL/ min. Calibration curve is determined using a series of calibration solutions of aflatoxin B1 in acetonitrile. The concentrations of calibration solutions may be 0.5, 1.5, 2.5, 5.0, 10.0, and 15.0 µg/ L. The retention time for aflatoxin B1 is near to 5.36 minutes or may

Aflatoxin B1 peak is identified in derivatized extract chromatogram by comparing its retention time with corresponding peak in the standard chromatogram. The quantity of the aflatoxin B1, '*C'* is determined in the derivatized extract (injected) from the respective standard curves. The concentration of aflatoxin B1 is calculated in test sample

Aflatoxins B1 ng/g = *C*/*W*

*W* = 50g × (2mL/ 200mL) × (0.02mL/ 2mL) = 0.005g Where *W* = equivalent weight of test portion (in 20µL) injected into LC; *C* = ng aflatoxin (in

**4.3.1 Extraction and clean-up procedure** 

**4.3.3 LC Determination with fluorescence detection** 

be slightly different by changing conditions or instrument.

evaporated under nitrogen.

**4.3.2 Aflatoxin derivatization** 

onto LC column.

**4.3.4 Calculations** 

20µL) injected into LC.

as follows:

#### **4.4 Determination of aflatoxin M1 in cheese by ELISA**

An ELISA method for the determination of aflatoxin M1 in cheese is given here as described by the protocol provided with RIDASCREEN® ELISA kit (RIDASCREEN® Aflatoxin M1 30/15, 2007).

#### **4.4.1 Sample preparation**

Cheese (2.0g) samples are first of all triturated. Extraction is completed with 40 mL dichloromethane by shaking for 15 minutes. The suspension is filtered and 10 mL of the extract is evaporated at 60°C under weak N2-stream. The oily residue is re-dissolved in 0.5 mL methanol, 0.5 mL PBS buffer and 1 mL n-heptane. After mixing thoroughly, it is centrifuged for 15 minutes at 2700 × *g*. The upper heptane layer is removed completely. From the lower methanolic-aqueous phase, 100µL is taken and diluted with 400 mL buffer 1 and 100µL of it is used per well in the test.

#### **4.4.2 Test procedure**

The standard solutions (100µL) and prepared samples (100µL) are added into the microtiter well placed in the microwell holder. Gentle mixing is accomplished by shaking the plate manually and incubated for 30 minutes at room temperature (20-25°C) in the dark. The liquid is poured out of the wells and microwell holder is tapped vigorously upside down against adsorbent paper to ensure complete removal of liquid from the wells. The wells are washed by adding 250 µL washing buffer in each well and poured out the liquid again. Washing step is repeated for two times. Then 100 µL of the diluted enzyme conjugate is added and mixed gently by shaking the plate manually and incubated for 15 minutes at room temperature in the dark. After incubation the wells are washed again. The 100 µl of substrate/chromogen is added and mixed gently by shaking the plate manually and incubated for 15 minutes at room temperature in the dark. Now stop solution (100µL) is added in each well. Mixing is done by shaking the plate manually. The absorbance is measured photometrically at 450 nm against an air blank with in 15 minutes after the addition of stop solution.

#### **4.4.3 Calculations**

The following formula is used to measure the % absorbance.

(Absorbance of standard or sample / absorbance of zero standard) × 100 = % absorbance The zero standard is made equal to 100 % and absorbance values are taken in percentages. A calibration curve is obtained by plotting %absorbance values for the standards against the aflatoxin M1 concentration (µg/L). The concentration of aflatoxin M1 in samples is calculated from the calibration curve.
